The invention relates to an apparatus and method for improving the speed and efficiency in performing thin layer chromatography (TLC) tests. Thin layer chromatography, one of the most standard laboratory techniques, is a solid-liquid partitioning technique used in chemical analysis. It is a micro-scale technique since only micrograms of material are needed to perform the analysis. A thin layer chromatography plate consists of a thin layer of adsorbent material (stationary phase) coated on a backing support. Typically, the support is, but is not limited to, a glass plate and the adsorbent material is, but is not limited to, a silica/silicate material. When the thin layer plate is partially submerged in a liquid (mobile phase), the liquid ascends the adsorbent by capillary action. By placing a small spot of a solution containing two compounds near the base of the thin layer plate, yet not immersed in the solvent, the ascending liquid carries the sample and partitions the sample between the adsorbent stationary phase and the liquid mobile phase (commonly referred to as developing). After development, the plate can be visualized and the distance a given compound travels with respect to the distance the solvent travels is referred to as the compounds Rf value and is expressed as a ratio. The Rf value is determined by a number of factors, namely the chemical structure of the compound and its interactions with the prescribed stationary and mobile phases. Changing the composition of either the stationary phase compound or mobile phase solvent can have dramatic effects on not only how far the sample travels with respect to the solvent front (Rf), but how tightly the compound travels together (the spot size and shape).
Different compounds have different interactions with the prescribed mobile and stationary phases and have a unique Rf of their own. Hence, thin layer chromatography is useful in determining the number of compounds in a given mixture. If the compounds in the mixture have very different chemical structures and the mobile/stationary phases are chosen properly, upon elution (development) the compounds will separate into their own spots with visible Rf differences between the spots. However, if the compounds have very similar chemical structure and/or the mobile/stationary phases are chosen poorly, the compounds may barely separate, if at all.
When ascertaining the chemical purity of a sample by TLC, the spot shape or the Rf difference between spots is not that crucial as long as all of the components of the mixture can be visualized. However, when one wants to physically separate the components of the mixture, the spot shape and the Rf difference between spots are crucial criteria when transferring what has been learned by TLC to the more macro-scale (mg-Kg) separation technique of column chromatography.
Column chromatography consists of a column, usually glass, a mobile phase, and a solid phase just as in thin layer chromatography. The adsorbent is packed into the column and the sample is loaded on top of the adsorbent. The mobile phase is then applied to the top of the column and flows through the column by either gravity or slight positive pressure. As in TLC, the column chromatography sample is partitioned between the mobile and stationary phases and depending upon the chemical structure of the compound, and the consistency of the mobile and stationary phases, the compound traverses down the column in a band. When a mixture of compounds is applied to the top of the column, and the proper mobile and stationary phases are chosen, as the solvent flows through the column the compounds separate into bands which can be isolated into pure compounds by collecting the bands as they elute out of the bottom of the column, commonly referred to as collecting fractions. When the consistency of these fractions is to be ascertained, one puts a spot from each fraction on a TLC plate and elutes (develops) with the same mobile and stationary phases as were run in the column. The factors that contribute to how well the compounds separate, are the volume of the fractions taken, the (low rate of the mobile phase, and most dramatically, the distance between the edges of the bands which is determined by the consistency of the mobile stationary phases. When the distance (between band edges) is large or when the bands are narrow, good separation can be achieved. Fractions 1-3 (figure not shown) can be combined and contain only pure compound A, fractions 4-7 (figure not shown) can be combined and contain only pure compound B, fractions 8-9 (figure not shown) can be combined and contain only pure compound C, and fractions 10-11 (figure not shown) can be combined and contain only pure compound D (Once compounds are pure, their structure can be elucidated by other spectroscopic means). However, when the distance (between band edges) is small or when the bandwidths are large, there can be a good amount of overlap in the fractions, such that only a few fractions contain pure compound. The other fractions either have to be thrown out, or resubmitted to another column chromatographic separation, which makes structure elucidation cumbersome if not impossible. This outcome is very undesirable from a synthetic standpoint, because it results in a loss of chemical yield and purity, and also a large loss of time if the mixtures have to be resubmitted to another column chromatographic separation. In all, poor separation is a very costly outcome.
The secret to separating mixtures of compounds using column chromatography rests in the ability to maximize the distance between bands and also minimize the width of the bands. The two greatest factors that influence this separation arc the consistency of the mobile and stationary phases. Since the typical number of stationary phases is limited to typically three or four substances, the chemist has at his disposal many pure mobile phases and an unlimited number of solvent combinations. However, one skilled in the art learns to get a feel of how to limit this number through trial and error and also prior experience. By testing a small number of solvents or combinations on TLC plates, using trial and error and experience, one can invest a tremendous amount of time trying to find an adequate mobile phase, since this is usually done in a linear stepwise non-scientific manner. This method often works, but this limited exploration and personal bias often lead to a solvent system that gives non-optimal separation between spots or large spot diameters, which when transferred to a column chromatographic separation often results in band overlap which reduces chemical purity and yield. This old method, as previously stated, is extremely undesirable especially if the compound being purified is extremely expensive and any loss of yield or purity is a loss of money.